This invention relates to a lignin-modifying white-rot fungus, Flavodon flavus (K1) Ryv., deposited at National Institute of Oceanography, Dona Paula, Goa, India, at accession No. NIOCC #312, and a process for removal of dyes in dye containing waste-waters and soil. The present invention particularly relates to a process for removal of synthetic dyes in dye containing waste-waters and the soil using lignin-modifying white-rot fungus, Flavodon flavus (K1) Ryv., NIOCC isolate 312. The lignin-modify white-rot fungus, Flavodon flavus (K1) Ryv. was also deposited at Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, U.S.A., bearing accession No. NRRL 30302 on Mar. 10, 2000.
Textile and dyestuff industries are the major contributors to industrial pollutants containing dyes. These dyes are highly stable in light and are also resistant to microbial attack. Most of these dyes are azo dyes and under anaerobic conditions, the azo linkages are reduced to form aromatic amines and these are toxic and carcinogenic (Meyer, U. 1981. Biodegradation of synthetic organic colorants. Federation of European Microbiological Societies Symposium. 12: 371-385). Due to the importance attached to prevention of environmental pollution, environmental agencies all over the world are imposing strict regulations for mitigation of pollution from industries. The effluents from the textile industries containing fast colored dyes are a major source of concern for environmentalists since such dyes besides causing aesthetic damage to sites, are toxic and carcinogenic. Industrial effluents from textiles, paper and pulp industries and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineering-Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). Remediation of such dye containing waste-waters using biological methods is termed bioremediation.
Normally, the textile dye waste-waters disposal includes physical-chemical treatment, waste minimization and biological treatment. Biological treatment includes biological pretreatment with activated sludge of textile waste-waters, and treatment in stabilization ponds (Groff, K. A. 1992, Textile waste. Water-Environment Research, 64: 725-729.). Unfortunately, waste-water treatment facilities are often unable to completely remove commercial dyestuff from contaminated waters and thus contribute to pollution of aquatic habitats. Some of these synthetic dyes are carcinogenic and are suggested to be responsible for tumor growth in some species of fish (see Bumpus J. A., B. J. Brock. 1988. Biodegradation of crystal violet by the white-rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology, 54:1143-1150).
Various organisms have been tried for degradation of dyes in textile waste-waters and bioremediation.
(i) Reference is made to the Japanese patent JP 06047394 Titled: Organic dye degradation in waste-water, issued on: 22.02.94, wherein a green alga Chlorella vulgaris is used for degradation of methylene blue by irradiating a microalga fermentor to generate OH radicals which in turn help in degradation of the dye. The method has a disadvantage as it involves irradiating the fermentor containing microalga and thus becomes expensive.
(ii) Reference may be made to U.S. Pat. No. 5,091,089 dated Feb. 25, 1992 Title: Decolorization of dye-containing waste-water, wherein, living, dead, free, immobilized white-rot fungi Myrothecium or Ganoderma sp. have been employed for adsorption, dye degradation and color removal. These fungi have not been tested for their efficiency in color removal in the presence of sea salts.
(iii) Reference may be made to U.S. Pat. No. 5,755,514 dated Mar. 24, 1992 Title: Increasing biodegradability of xenobiotic azo dyes wherein, white-rot fungus Phanerochaete chrysosporium and actinomycetes Streptomyces spp are used in degradation of xenobiotic azo dyes. They are not shown to degrade azo dyes in the presence of sea salts.
(vi) Reference may be made to a publication wherein Phanerochaete chrysosporium is reported to degrade textile azo dyes very efficiently under conditions where lignin-modifying enzymes are produced by incubating cultures at 39xc2x0 C., (Capalash, M. and P. Sharma. 1992. Biodegradation of textile azo-dyes by Phanerochaete chrysosporium. World Journal of Microbiology and Biotechnology. 8:309-312). However, it has not been shown to degrade synthetic dyes in the presence of sea salts. The fungal culture needs to be incubated at 39xc2x0 C. for effective degradation, which may involve additional step during treatment of wastewater.
(v) A reference may be made to German patent (DD-290004, entitled xe2x80x98Microbial breakdown of xenobiotic dyes of triphenylmethane seriesxe2x80x99, issued on May. 16, 1991) wherein, degradation of crystal violet and malachite green are brought about by oleophilic Gram-positive bacteria preferably Corynebacterium sp. IMET 11347 or Mycobacterium sp.
IMET 11349. The disadvantage of this system is that the organisms have to be grown at 32xc2x0 C. in 1% methanol and removal of bacterial inoculum from dye-containing waste-water will not be very easy.
The fungus Flavodon flavus belonging to the class Basidiomycetes produces fertile basidiomata in medium containing alpha-cellulose and sometimes in malt extract agar medium on prolonged incubation. Most of the times the fungus is in non-sprouting form and can be recognized by crystals deposited around fungal hyphae.
Many lignin-degrading fungi can also degrade textile azo dyes but their growth and enzyme production in presence of synthetic sea water has not been demonstrated. The applicants have used a strain of Phanerochaete chrysosporium and observed that it does not grow in synthetic sea water. This implies that it cannot effect dye degradation in the presence of sea water either. Caplash et al., 1992 have also used a strain of Phanerochaete chrysosporium in their studies.
Although presence of lignin-modifying enzymes are reported in several fungal taxa belonging to the class Basidiomycetes, the applicants reported their presence in Flavodon flavus for the first time (Raghukumar, C., T. M. D""Souza, R. G. Thom and C. A. Reddy, 1999. Lignin-modifying enzymes of Flavodon flavus, a basidiomycete isolated from a coastal marine environment. Applied Environmental Microbiology 65:2103-2111). Flavodon flavus, NIOCC isolate #312 is a close relative of Irpex and the Polyporus-Trametes lineage of polypores (Ryvarden L. 1991. Genera of Polypores. Synopsis Fungorum 5, Fungiflora, Oslo, Norway). Cultural characteristics of the isolate #312 were studied in malt extract agar medium and on alphacel agar. Fertile basidiomata that occurred on this media were identified as Flavodon flavus on the basis of their smooth nonamyloid basidiospores, dimitic hyphal system with skeletal hyphae and simple septet generative hyphae, incrusted hymenal cystidia and poroid-hydnoid yellow fruiting bodies. Occurrence and taxonomy of this fungus is reported from Indian forests (Sen M. 1973. Cultural diagnosis of Indian Polyporaceae. 3. Genera Daedalea, Favolus, Ganoderma, Hexagonia, Irpex, Lenzites, Merulius, and Poria. Indian Forest Records (New Series) Forest Pathology, Vol. 2, No. 11, Dehra Dun, India). The applicants have isolated it from marine habitat for the first time. The applicants also shown that it grows much better in the presence of synthetic sea water. (The identification of this fungus was carried out by Dr. R. G. Thorn from Department of Botany, University of Wyoming, Laramie, WY 82071-3165, U.S.A., who is also a co-author in the above mentioned publication).
The main objective of the present invention is to identify and provide novel white-rot fungus Flavodon flavus (K1) Ryv. Deposited at National Institute of Oceanography, (and at Agricultural Research Service Culture Collection (NRRL), 1815 North University Street, Peoria, Ill. 61604, U.S.A., bearing accession No. NRRL 30302), exhibiting lignin degrading properties.
Another object of the invention is to provide a process for removal of dyes using the lignin-modifying white-rot fungus, Flavodon flavus, for possible use in dye-containing waste-water and in saline soils. The said fungus can be efficiently utilized for the above-mentioned usage in fresh water as well as under estuarine conditions because of its tolerance to sea salts.
Yet another objective is to provide a process for cultivation of the novel fungus on a large scale using inexpensive raw material such as sugarcane bagasse suspended in distilled water or 50% artificial sea water or simple medium like malt extract broth prepared with fresh water as well as 50% artificial sea water.
In accordance with the foregoing objects, the invention provides novel white-rot fungus Flavodon flavus deposited at National Institute of Oceanography, Dona Paula, Goa, India, at accession No. 312. The invention also provides a process for removal of dyes in dye-containing effluent waters and soil, which comprises the steps of growing the white rot fungus Flavodon flavus in a nutrient medium containing assimilable carbon and nitrogen source, having optimal salinity up to 15 parts per thousand for a period of about 4-10 days, contacting biomass with effluents containing dyes for a period of at least 5 days followed by separation of the fungal biomass from the effluents by conventional methods to render the effluents substantially free of dyes.
In an embodiment, the fungus F. flavus is capable of treating estuarine waters, fresh water, effluents selected from textile, leather and paper industries.
In another embodiment, the effluents may be waste-water containing dyes or soil containing dyes.
In an embodiment the nutrient medium used for growth of the fungus is selected from glucose, sugar-cane bagasse suspended in distilled water, synthetic media, low. nitrogen medium, malt extract broth prepared with fresh water and 50% sea water.
In another embodiment, the fungus F. flavus is grown in nutrient medium comprising malt extract broth containing about 2% malt extract and about 0.3% peptone in distilled water.
In yet another embodiment, synthetic media is prepared in distilled water or 50% artificial sea water containing 1% glucose as carbon source, 2.4 mM ammonium tarterte as nitrogen source, thiamin, trace metal solution, macro element solution containing potassium and manganese salts, Tween 80, veratryl alcohol and 20 mM sodium acetate buffer at pH 4.5.
In a further embodiment, the low nitrogen medium comprises 10 g glucose as the carbon source, 2.4 mm ammonium tartrate as the nitrogen source, 70 ml of trace metal solution from stock solution containing MgSO4 3 g, MnSO4 0.5 g, NaCl 1.0 g, FeSO4 7H2O 0.1 g, CoCl2 0.1 g, ZnSO4 7H2O 0.1 g, CuSO4 0.1 g, AlK(SO4), 12H2O 10 mg, H3Bo3 10 mg, Na2MoO4 2H2O 10 mg, Nitrilotriacetate 1.5 g in 1 L distilled water, one hundred ml of macro element solution containing KH2PO4 20 g, MgSO4 5 g, CaCl2 1 g in 1 L of distilled water, one percent Tween 80, 1 ml from 0.4 M stock solution of veratryl alcohol, 10 ml of thiamin from stock solution containing 100 mg in 1 L distilled water and 20 mm sodium acetate buffer.
In yet another embodiment, the pH of the nutrient medium is maintained at 4.5.
In a further embodiment, the salinity of the medium for growing the fungus is maintained at about 0 to 15 parts per thousand.
In yet another embodiment, the fungus is grown in the nutrient medium for a period of about 4-10 days.
In another embodiment, the fungal biomass is immobilized on a conventional support such as nylon mesh by conventional methods selected from immobilization and adsorption.
In yet another embodiment, the fungus is capable of degrading pollutants selected from the group of synthetic dyes comprising azo, heterocyclic and polymeric dyes.
In a further embodiment, the effluents are degraded by contacting with the effluents with fungal biomass.
In yet another embodiment, the fungal biomass is separated from the effluents manually or by filtration.
In an embodiment of the present invention, a process is provided for removal of synthetic dyes using the lignin-modifying white-rot fungus, Flavodon flavus, NIOCC 312 for possible use in dye-contaminating waste-waters and soil in the presence of salts. The said fungus may be efficiently utilized for the above-mentioned usage in fresh water as well as under estuarine conditions because of its tolerance to sea salts.
In another embodiment of the present invention, the fungus may be grown on large scale in an inexpensive raw material such as sugarcane bagasse suspended in distilled water or 50% artificial sea water or simple medium like malt extract broth prepared with fresh water or with 50% artificial sea water. The biomass of the fungus thus raised may be used for seeding dye-contaminated wastewater in a freshwater or estuarine environment or for seeding soil contaminated with synthetic dyes.
In yet another embodiment, the said fungus may be immobilized on a suitable substrate, and the immobilized fungus may be used for seeding the soil and aquatic habitats contaminated with synthetic dyes.
The organism given in the present invention is a white-rot basidiomycete fungus isolated from a decaying marine plant from a coastal marine environment and identified as Flavodon flavus. The said fungus F. flavus can be grown in malt extract broth containing 2% malt extract and 0.3% peptone in distilled water. The biomass of the fungus can be used for seeding soil contaminated with synthetic dyes for treating dye-containing waste water under normal conditions or under estuarine conditions. The biomass of the said fungus thus raised can also be immobilized using conventional methods used for immobilizing fungi and used for bioremediation of dye-containing aquatic systems.
The fungal mat is macerated using a homogeniser and used as starter inoculum for the experimental cultures of synthetic media prepared in distilled water or in 50% artificial seawater. The synthetic media can be prepared in distilled water or 50% artificial sea water containing 1% glucose as carbon source, 2.4 mM ammonium tartrate as the nitrogen source, thiamin, trace metal solution, macro element solution containing potassium and manganese salts, Tween 80, veratryl alcohol and 20 mM sodium acetate buffer at pH 4.5. This medium is referred to as low nitrogen medium. An example of the process for dye degradation involves addition of various synthetic dyes such as Azure B. Brilliant Green, Congo Red, Crystal Violet, Remazol Brilliant Blue R and polymeric dyes such as Poly B-411 and Poly R-478 at a final concentration of 0.02% to 3 day old cultures of F. flavus growing in malt extract broth, synthetic medium prepared with distilled water or 50% artificial sea water. The degradation of dyes can be monitored spectrophotometrically by removing an aliquot of sample from these cultures and measuring changes in absorbance at respective wavelengths of various dyes every alternate day up to 10 days. Heat-killed cultures serve as controls where no decolorization or degradation of dyes is observed. Moreover, the said fungus can be grown on a large scale using an inexpensive raw material such as sugarcane bagasse suspended in distilled water or in 50% artificial sea water. The said fungus produces lignin-modifying enzymes such as manganese-dependent peroxidase (MNP), lignin peroxidase (LIP) and laccase, in conventional media prepared with distilled water as well as in media prepared with 50% artificial sea water.
The fungus can be grown in the medium generally referred to as low nitrogen medium (Tien M. and T. K. Kirk, 1988. Lignin peroxidase of Phanerochaete chrysosporium. Methods in Enzymology 161: 238-249). The low nitrogen medium contains 10 g glucose as the carbon source, 2.4 mm ammonium tartrate as the nitrogen source, 70 ml of trace metal solution from stock solution containing MgSO4 3 g, MnSO4 0.5 g, NaCl 1.0 g, FeSO4 7H2O 0.1 g, CoCl2 0.1 g, ZnSO4 7H2O 0.1 g, CuSO4 0.1 g, AlK(SO4), 12H2O 10 mg, H3Bo3 10 mg, Na2MoO4 2H2O 10 mg, Nitrilotriacetate 1.5 g in 1 L distilled water. One hundred ml of macro element solution containing KH2PO4 20 g, MgSO4 5 g, CaCl2 1 g in 1 L of distilled water. One percent Tween 80, 1 ml from 0.4 M stock solution of veratryl alcohol, 10 ml of thiamin from stock solution containing 100 mg in 1 L distilled water and 20 mm sodium acetate buffer to maintain pH at 4.5. the culture needs to be flushed with pure oxygen every third day as it is known that lignin-degrading enzyme system works well under conditions of oxygen saturation.
Low nitrogen medium prepared with half strength synthetic seawater for growing this fungus has been reported for the first time by the applicants. This modification of low nitrogen medium of Tien and Kirk (1988) was optimized by the applicants.
Cultural characters of #312 were studied in malt extract agar medium (MEA) containing 1.25% malt extract and 2% agar (Nobles, 1948). The culture code provided for #312 is that of (Nobles, 1965; Ginns and Lefebvre, 1993); 2a.6.8.13.32.36.40.43.48.50.54, where code 2a represents detection of laccase with a positive syringaldazine spot-test and peroxidase as detected by application of 0.4% H2O2 to a syringaldazine spot-test (Harkin and Obst, 1973 . Growth fast, plates covered in 2-3 weeks (code 42-43); margin even, appressed, fimbriate to wispy, hyaline, aerial mycelium sparse to moderate after 2 weeks, low and thin, downy with cottony patches, hyaline to white; after 6 weeks mostly cottony, white to cream with sulfur yellow patches; reverse of the plates bleached (code 40); not fruiting within 6 weeks (code 48); odour nil to faintly resiny-fragrant (code 50); marginal hyphae tubular, straight, thin-walled, 2.5-4.0 xcexcm diam, with simple septa, sparsely branched, mostly from immediately behind septa; dolipore septa visible with ammoniacal congo red; cultures monomitic but in age with scattered, encrusted cystidia resembling skeletal hyphae; lacking any form of conidia or noteworthy hyphal swellings.
Several very old subcultures fruited on MEA and alphacellulose agar producing identifiable basidiomata. Basidiomata pulvinate, 2-5 mm broad, sulphur yellow, irregularly poroid-toothy; pores approximately circular, 1-2 per mm, lined with a hymenium of 4-spored, clavate basidia (24-28xc3x976-8 xcexcm) and fusoid-cylindric, thick walled cystidia (28-38xc3x973.5-5.0 xcexcm). The hyphal system was dimitic with simple septate generative hyphae 2.0-3.5 xcexcm in diam and infrequently branched, tubular skeletal hyphae 2.0-5.0 xcexcm in diam. Skeletal hyphae terminated in the hymenium as cylindrical, thick-walled pseudocystidia. Basidiospores hyaline, nonamyloid, smooth, thin-walled, elliptical to lacrymoid, 6.0-7.0xc3x973.0-3.8 xcexcm. Fruiting bodies from culture were identified as Flavodon flavus (Klotzsch) Ryvarden using the keys in Ryvarden and Johansen (1980).
Lack of salt tolerance in most ectomycorrhizal basidiomycetes was reported by Hutchinson (1990) and in wood-inhabiting corticoid fungi by Thorn (1991). Tolerance to synthetic sea salt as shown by growth, production of lignin-modifying enzymes and dye decolorization in this trail of F. flavus was reported for the first time by the applicants (Raghukumar et al., 1999).
The said fungus Flavodon flavus, NIOCC isolate 312, is capable of growing in the presence of salts whose concentration is similar to that found in half-strength sea water. Most of the industrial effluents from textiles, dyestuff, paper and pulp and leather industries contain chromogenic substances as well as high concentrations of salts, especially chlorides and sulfates (Public Health Engineeringxe2x80x94Design in Metric waste-water treatment by R. E. Bartlett, 1971, Applied Science Publishers Ltd., London). In light of this, salt tolerant organisms are better suited for such waste-water treatments.: Most of the fungi used for bioremediation of such colored waste-waters have not been tested for their salt tolerance. In view of this, the present process has an advantage over the conventional processes referred to in various patents discussed above. White-rot fungi are unique among eukaryotic microbes in possessing powerful lignin-degrading oxidative enzymes such as MNP, LIP and lacasses, which have a broad substrate specificity and are thus able to oxidize several environmental pollutants. Results from several laboratories have shown that the ability of white-rot fungi such as Phanerochaete chrysosporium and Trametes versicolor to degrade an array of pollutants including synthetic dyes such as axo, heterocyclic and polycyclic dyes is due to the lignin-modifying/degrading system (C A. Reddy. 1995. The potential for white-rot fungi in the treatment of pollutants. Current Opinion in Biotechnology, 6: 320-328). The fungal isolate F. flavus, NIOCC isolate 312, obtained from marine environment also produces lignin-modifying enzymes such as MNP LIP and laccase. It produces these enzymes in natural media such as ME broth, conventional synthetic medium prepared with distilled water or 50% artificial sea water and also in powdered sugarcane bagasse suspended in distilled water or in 50% artificial sea water, and degrades synthetic dyes such as Azure B, Brilliant Green, Crystal Violet, Congo Red, Remazol Brilliant Blue R and polymeric dyes such as Poly B and Poly R.
Thus, this invention particularly relates to degradation of synthetic dyes in the presence of salts by the fungus F. flavus deposited at the National Institute of Oceanography, Dona Paula, Goa 400004, India, having accession number NIOCC 312, in textile waste-water treatment, and bioremediation of soil. The said fungus can also be grown in conventional media or in powdered sugarcane bagasse suspended in distilled water or 50% artificial sea water to raise large biomass of the fungus for application in field trials for bioremediation in the presence of sea water or fresh water. The said fungus thus grown can be immobilized by conventional methods and used for removal of various synthetic dyes from contaminated soil as well as aquatic habitats. The said fungus, F. flavus produces lignin-modifying enzymes such as manganese-dependent peroxidase E. C. 1.11.1.7 (NNP), lignin peroxidase, E. C. 1.11.1.7 (LIP) and laccase, E. C. 1.10.3.2 when grown on sugarcane bagasse suspended in plain distilled water or in 50% artificial sea water or in conventional media prepared with distilled water or 50% artificial sea water. By virtue of these lignin-modifying enzymes which break down a broad range of polymeric substrates, this fungus is useful in degradation of pollutants such as synthetic dyes. Thus degradation of synthetic dyes in waste-waters can be achieved by using the fungus F. flavus in fresh water as well as estuarine conditions.